Quantitative End-group Analysis Research Articles

A qualitative and quantitative post‐mortem analysis: Studying free‐radical initiation processes via soft ionization mass spectrometry

AbstractThe current article contains a review of the electrospray ionization‐mass spectrometry characterization of polymers prepared via thermal‐ and photoinitiation processes. The used analysis method permits direct access to detailed endgroup information. For a qualitative and quantitative endgroup analysis, sophisticated methods have been developed which provide a detailed image of the incorporation propensity of thermally as well as photolytically generated radicals at the polymer chain termini. Such a post‐mortem analysis of polymeric materials specifically allows for the quantification of the ability of radical fragments to initiate polymerization processes. Herein, the most recent progress in the field of mass spectrometric radical reactivity mapping is outlined and open questions as well as future directions are discussed. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Polymerization of epoxides catalyzed by a mixed-valent iron trifluoroacetate [Fe3O(O2CCF3)6(H2O)3

The readily available mixed-valent iron trifluoroacetate complex [Fe2IIIFeII(µ3-O)(O2CCF3)6(H2O)3] is an effective catalyst for the polymerization of epoxides. A very small amount of the catalyst (1.0–0.01 mol%) could initiate the polymerization of cyclohexene oxide, cyclopentene oxide and epichlorohydrin. Based on quantitative end-group analysis by 19F NMR spectroscopy, a Lewis acid (LA⊕) catalyzed anionic reaction mechanism is proposed. Copyright © 2012 Society of Chemical Industry

Number-average molecular weight of radiation-degraded poly(tetrafluoroethylene). An end group analysis based on solid-state NMR and IR spectroscopy

Changes in the chemical structure of poly(tetrafluoroethylene) (PTFE) induced by electron beam irradiation at room temperature in air have been studied by 19F solid-state NMR and IR spectroscopy. Chain scission associated with the formation of trifluoromethyl (–CF 3), carboxylic acid fluoride (–COF) and carboxylic acid (–COOH) end groups was confirmed to be the predominant process under these conditions. The number-average molecular weight of radiation-degraded PTFE as a function of irradiation dose was calculated based on quantitative end group analysis. It strongly decreases with increasing dose to approximately 4000 g/mol at a irradiation dose of 4 MGy. The molar ratio of CF 3 end groups to oxygen-containing (–COF, –COOH) end groups was found to depend on the irradiation dose and the irradiation conditions. For low dose level, the molar ratio determined experimentally coincides well with the mechanism proposed for radiation-induced degradation of PTFE.

Structural Characterization of Aromatic Polyester Resins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize average molecular weights and end-groups of synthetic aromatic polyesters and the same characteristics were also determined for acetylated polyester samples. In addition, the progress of a polyesterification reaction was followed by mass spectrometry by investigating samples taken from the reaction mixture at different reaction times. Average molecular weights were compared with those measured by gel-permeation chromatography (GPC) and the results of quantitative end-group analysis were considered with the acid values of the polyesters. MALDI measurements were carried out using two different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 2-(4-hydroxyphenylazo) benzoic acid (HABA). Different matrices and derivatization of the polyester influenced the width of the polymer distribution determined by mass spectrometry and further affected the proportions of differently terminated oligomers, in addition to the average molecular weight. The relative amount of the most abundant oligomer species was increased when the polymer distribution width was increased.

Qualitative and quantitative end-group analysis of a small molecular weight polyester by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for qualitative and quantitative end-group analysis of a small molecular weight polyester, poly(2-butyl-2-ethyl-1,3-propylene phthalate). The presence of carboxyl-terminated linear and cyclic polyester oligomers was confirmed with the help of simple sample preparation methods. The presence of carboxyl end-groups in the polyester chains was verified through their formation of carboxylate salts with alkali metal cations. Cyclic oligomers were identified through deuterium exchange of the exchangeable protons of the polyester. Various inorganic salts were tested for salt formation of the carboxyl end-groups, but only the alkali metal salts proved effective. The influence of the alkali metal salts on the results of the quantitative end-group analysis was also studied. The relative amounts of differently terminated and cyclic oligomers were calculated when the alkali metal salts were used with different matrices. The results showed that both the salts and the matrices used in sample preparation can have a marked effect on the quantitative results of the end-group analysis. The measurements were carried out using 2,5-dihydroxybenzoic acid (DHB), 1,8, 9-trihydroxyanthracene (dithranol), and 2-(4-hydroxyphenylazo)benzoic acid (HABA) as matrix compounds. Dithranol and HABA repeatably exhibited similar results, and these results differed from those obtained with DHB probably because of the different ionization mechanisms in the MALDI process.

Evidence for a diprotomeric structure of Na,K-ATPase: Accurate determination of protein concentration and quantitative end-group analysis

Three methods were used to assess protein concentration in membrane-bound Na,K-ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH 2-terminal residues of the β-subunit (NH 2-Ala) and one such residue of the α-subunit (NH 2-Gly) per ligand-binding unit. From the data on equimolarity of the α- and β-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two αβ-protomers, one possessing a modified (presumably an N-blocked) α-subunit.

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH 2-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.

Studies on Yeast Enolase: QUANTITATIVE END GROUP ANALYSES AND THE EFFECT OF EXOPEPTIDASE DIGESTION

Abstract The structural model of yeast enolase has undergone substantial modification in recent years, and the quantitative end group analysis of the enzyme was undertaken as a means of establishing the most recent two-subunit model by a chemical method. Carboxypeptidase digestion and hydrazinolysis gave 1.95 and 1.85 moles, respectively, of carboxyl-terminal leucine per mole of enzyme, and amino-terminal analysis by the cyanate method gave 1.8 moles of amino-terminal alanine per mole of enzyme, thus confirming the model of yeast enolase as an 88,000-dalton protein consisting of two 44,000-dalton subunits. Contrary to reports in the literature, pure enolase was found to be very resistant to endopeptidase-free exopeptidase digestion. The earlier findings that large segments of both the amino-terminal and carboxyl-terminal sequences can be removed by exopeptidases without loss of enolase activity can thus not be reproduced in this laboratory. Some possible reasons for this discrepancy are discussed.

Structural Studies on Bovine Lactoferrin

Abstract Bovine lactoferrin has a molecular weight of 77,100 ± 1500 in dilute aqueous solution as determined by the method of sedimentation equilibrium in the ultracentrifuge. In 6 m guanidine hydrochloride in the presence or absence of mercaptoethanol, bovine lactoferrin has a molecular weight of 72,500 to 77,200 ±1300 as measured by sedimentation equilibrium in the ultracentrifuge or by gel filtration on agarose columns. The molecular weight obtained by gel electrophoresis in the presence of sodium dodecyl sulfate is 76,000 ± 2400. Tryptic peptide maps reveal a number of peptides approximately equal to the content of lysine plus arginine in the molecule. Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain. Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin. Another protein, isolated as a major contaminant in commercial preparations of bovine α-lactalbumin showed identical properties to bovine lactoferrin except that it appeared, based upon molecular weight measurements, to be an aggregating system.

The structure of tobacco necrosis virus: II. Terminal amino acid residues of the protein subunit

The amino end group of the protein of tobacco necrosis virus (TNV), unlike those of most other plant viruses, was found to be unsubstituted. Reactions with fluorodinitrobenzene and 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl) resulted in the identification of alanine as the amino terminal residue. Leucine aminopeptidase, at slightly alkaline pH, released up to 62% of the theoretical value of alanine from the whole virus. The TNV coat protein N-terminus is therefore identical to that of its satellite and the RNA-containing bacteriophages. Kinetic studies with carboxypeptidase suggested that the C-terminal sequence of tobacco necrosis virus coat protein is …Ser·Val·Val·Met. Quantitative end-group analysis was in agreement with the earlier suggested molecular weight of the protein subunit as 33,300. The number of tryptic peptides separated on fingerprints was also consistent with these data.

Physikalische, chemische und biologische Charakterisierung von menschlichem Choriongonadotropin

Purification of commercially available HCG preparations with DEAE Sephadex A 50 and Sephadex G 100 column chromatography gave homogeneous fractions having specific biological activities four fold those of the starting materials. The purified HCG has ICSH characteristics and, in high doses, a definite FSH effect is present. Chemical analysis of HCG showed it to contain 29% carbohydrates and 69% peptides. The C-terminal amino acid of the peptide chain was found to be serine, but the N-terminal amino acid could not be determined with normal methods. A molecular weight of 22000 — 27000 daltons was obtained by quantitative end group analysis. Ultracentrifugation experiments in 4 m guanidine hydrochloride gave a molecular weight of 27200 daltons, but in neutral saline solutions at HCG concentrations above 2 mg/ml the apparent molecular weight was higher and indicated dimer formation. A dissociation constant of 10-5 mol/l was estimated for the monomer-dimer equilibrium. Since biological activity is found with 0.1 to 0.5 µg, it was concluded that the HCG monomer is the active entity. The purified HCG is stable from pH 4.5 to pH 10 for 6 hours at 37 °C. At pH 2.5 only 5 to 10% of the original activity is retained. HCG is rapidly inactivated at 100 °C, but a residual activity of 6 — 10% remained after 30 minutes at 80 °C. No activity was lost after 30 minutes incubation at 60 °C.